Notch signaling is essential for development of numerous tissues, and dysregulation of Notch activity results in a wide variety of diseases including several cancers (e.g. T-cell acute lymphoblastic leukemia), and developmental disorders such as Alagille Syndrome and congenital heart defects. Glycosylation of the Notch extracellular domain (ECD) provides a critical mechanism for regulating Notch activity. Loss of O-fucose or O- glucose glycans blocks Notch signaling, and extension of O-fucose by the Fringe family of 3-N- acetylglucosaminyltransferases modulates Notch specificity for ligand. The Notch ECD contains up to 36 tandems Epidermal Growth Factor-like (EGF) repeats, most of which are decorated with these glycans at predicted consensus sequences. Using cell-based Notch signaling assays to evaluate the importance of individual O-fucose and O-glucose modification sites in mouse Notch1, we identified two functional regions, the ligand-binding domain (EGF11-12) and the Abruptex region (EGF24-29), that mediate the effects of these glycans. Interestingly, the Abruptex region is known to be genetically linked to Fringe in flies. We also developed highly sensitive semi-quantitative nano-LC-MS/MS glycoproteomic methods to examine the structure of glycans at individual sites on Notch proteins overexpressed in cell-based systems. Significantly, O- fucose sites in the ligand-binding and Abruptex regions of Notch1 are modified at high stoichiometries and are efficiently elongated by Fringes. Finally, published structural studies and our preliminary electron microscopic structural studies suggest that glycan distribution and structure influence Notch conformation. Based on these observations, we have proposed that Notch1 function is regulated by glycan structures in the ligand- binding and Abruptex regions of the Notch1 extracellular domain. Here we will refine and test our model by examining whether glycosylation of the same regions also affect Notch2, which is essential for development but plays distinct non-redundant roles with Notch1. In Aim 1 we evaluate the effects of mutations in predicted O-fucose and O-glucose sites in mouse Notch2 using cell-based assays. If glycans regulate all Notch proteins through a common mechanism, we predict that elimination of glycosylation sites in the ligand-binding and Abruptex regions will affect Notch2 activity as they do in Notch1. Alternately, one or more of the glycosylation sites responsible for regulation may differ for Notch1 and Notch2. In Aim 2 we use our highly sensitive glycoproteomic methods to examine whether these functionally important sites are modified in vivo during development of B and T cells, where Fringes are known to modulate Notch activity. Finally, in Aim 3 we collaborate with several world-class structural biologists to determine how site-specific changes in glycans affect Notch structure and function. These studies will define the contribution of glycan distribution and structure to Notch function i vivo and will provide the foundation for future development of novel therapeutic strategies taking advantage of Notch regulation by glycosylation.